The coagulation of blood occurs through a complex series of reactions that function as a biological amplifier and culminate in the conversion of soluble circulating fibrinogen into a fibrin meshwork at the site of a vascular injury, providing stability to a hemostatic plug of platelets. In this system, relatively few initiating substances sequentially and proteolytically activate a cascade of circulating precursor proteins, zymogen clotting or coagulation factors. Among the reactions is the conversion of the zymogen, prothrombin, to the activated enzyme thrombin, which is the pivotal enzyme of the coagulation system. Thrombin is a serine protease that rapidly activates platelets, activates other clotting factors, and converts fibrinogen to insoluble fibrin. Thrombin also converts the zymogen FXIII to FXIIIa, which chemically cross-links the fibrin clot.
Abnormalities in the coagulation cascade can have potentially fatal effects, leading to extremes of bleeding disorders and excessive clotting, e.g. thrombosis. In addition, anticoagulant medications cause abnormalities in the coagulation cascade.
The coagulation system may be assessed by activating the cascade and measuring the time it takes for a blood or plasma sample to clot. Clotting times provide clinically useful information, however, they only represent the initial (<5%) thrombin generation. The majority of thrombin is formed after this initial period.
Attempts have been made to quantify the dynamics of thrombin formation. In one such method, a thrombin activator is added to a plasma sample together with a fluorogenic thrombin substrate. Thrombin formed during the clotting reaction consumes the substrate, producing a conversion product that is detected fluorometrically in real time. From these data can be calculated the endogenous thrombin potential (ETP, also referred to as the area-under-the-curve), which indicates how much thrombin has been active and for how long. The data can also be used to calculate lag time (the time to formation of thrombin), the maximal thrombin concentration reached, and the time to the peak thrombin formation. However, this method is unable to measure thrombin generation in whole blood, primarily due to fluorescence signal quenching by components in whole blood including red blood cells. A method to detect thrombin generation in whole blood was subsequently developed which included sequestering the fluorogenic product in a layer (filter paper) such that its fluorescence would not be quenched by red blood cells.
Francis et al., (WO2011094185) describe a method for measuring generation of thrombin in a sample of whole blood as a function of time. The method comprises adding to a sample of whole blood a small peptide fluorogenic substrate and a thrombin activator to form an activated sample. A conversion product is permitted to form in the activated sample. Fluorescence is measured as a function of time from a fluorescent group that is released during the formation of the conversion product with the use of a fluorescence detector. The fluorescence detector operates in an extended range mode and has increased sensitivity. Thrombin generation as a function of time can then be calculated from the measured fluorescence.
These methods of measuring thrombin generation are limited in that they detect free thrombin as well as thrombin that is bound to alpha-2-macroglobulin. In order to obtain an accurate measure of physiologically active thrombin in a blood sample, assays to measure thrombin generation must correct for thrombin bound to alpha-2-macroglobulin. While an assay method has been developed which does not measure thrombin bound to alpha-2-macroglobulin, for example as described U.S. Pat. No. 8,138,308 in which a polymer is attached to a fluorogenic thrombin substrate such that thrombin bound to alpha-2-macroglobulin cannot cleave the substrate, it would be desirable to develop an assay that measures activity or generation of an activated protease in the blood.